Cephalosporin derivatives

ABSTRACT

Process for the preparation of 7β-amino-3-substituted methyl-3-cephem-4-carboxylic acid derivatives, new intermediates comprising 7β-(cyclo)alkylideneammonio-3-halomethyl-3-cephem-4-carboxylic acid derivatives and 7β-amino/ammonio-3-bromomethyl-3-cephem-4-carboxylic acid derivatives and the processes for the preparation of these intermediates.

PRIOR APPLICATION

This application is a division of U.S. patent application Ser. No.216,877 filed Jul. 8, 1988, now U.S. Pat. No. 4,921,954.

The invention relates to a new process for the preparation of certaincephalosporins. More particularly, the invention relates to a one-potprocess for the preparation of 3-substitutedmethyl-3-cephem-4-carboxylic acid derivatives.

European Patent Application No. 0137534 discloses a multi-step one-potprocess for the preparation of 7β-amino- and 7β-acylamino-3-substitutedmethyl-3-cephem-4-carboxylic acid derivatives by a bromination of the3-methyl group of a deacetoxycephalosporin 1β-oxide, replacement of thebromo atom in the 3-bromomethyl group by another substituent,deoxygenation of the sulphoxy group and removal of a 7β-acyl substituentto give a cephalosporin with the free 7β-amino group. In this processthe introduction of new substituents leading to various importantintermediates takes place halfway the process. This makes optimizationnecessary for every single intermediate.

It has surprisingly been found that new nucleophilic substituents can beeasily introduced at the last stage of the process without theoccurrence of any side-reaction, such as isomerisation of the doublebond. This was unexpected because reactions of the cephalo-derivative inthe deoxygenated form generally are more sluggish and may give rise toisomerisation in the cephem moiety.

Therefore, in accordance with the present invention a process isprovided for the preparation of 3-substitutedmethyl-3-cephem-4-carboxylic acid derivatives of formula I ##STR1##wherein A is --NH₂, ##STR2## P is --COOH; or --COO.sup.⊖ when Q is --CH₂.sup.⊕ R₂,

Q is --CH₂ R₁ and A is NH₂ ; or Q is CH₂ X and A is --NH₂, NH₂.HX or##STR3## or Q is --CH₂ .sup.⊕ R₂ and A is --NH₂ ; or Q is --CH₂ .sup.⊕R₂.X.sup.⊖ and A is --NH₂ or --NH₂.HX,

R₁ is (lower)alkoxy, (lower)alkylthio, (lower)alkanoyloxy,(lower)alkanoylthio or S--R₃ where R₃ is an optionally substitutedheterocyclic ring,

X is halogen or a mixture thereof when A is ##STR4## or X is bromo orchloro or a mixture thereof when A is --NH₂ or --NH₂.HX,

R₂ is a ##STR5## R₄ is alkyl, with up to 8 carbon atoms, R₅ is alkyl,with up to 8 carbon atoms, whereby R₄ and R₅ are the same or differentor R₄,R₅ together with the carbon atom to which they are attached formcycloalkylidene with up to 8 carbon atoms;

starting from 3-methyl-3-cephem-4-carboxylic acid 1β-oxide derivativesof formula II, ##STR6## wherein B is ##STR7## T is --CH₃ when B is##STR8## or T is --CH₂ X where X is halogen or a mixture thereof when Bis ##STR9## or X is bromo or chloro or a mixture thereof when B is --NH₂or --NH₂.HX,

R₄ and R₅ are as defined above,

R₆ is an optionally substituted methyl group --CH₂ R₇ which can beintroduced by penicillin fermentation, and wherein R₇ is hydrogen, aryl,alkyl, cycloalkyl, alkenyl, aryloxy, alkyloxy, arylthio, alkylthio, orR₆ is an optionally substituted aryl group,

by carrying out subsequently the following reactions preferably in thesame reaction vessel without isolation of the intermediate products:

when A is --NH₂, Q is --CH₂ R₁, or CH₂ .sup.⊕ R₂ or --CH₂ .sup.⊕ R₂X.sup.⊖, and P, R₁₋₃ and X are as defined above, and

B is ##STR10## and T, R₆ and R₇ are as defined above:

a) silylation of the carboxy group, optionally carried out in situ,

b) light-induced bromination of the 3-methyl group of a compound offormula II to give a compound with the 3-bromomethyl group, using aN-bromo-amide or a N-bromo-imide as brominating agent,

c) if necessary, replacement of any bromine introduced in step b) in themethylene group adjacent to the sulphur atom in the dihydrothiazine ringby hydrogen by reaction with a trialkyl or triaryl phosphite,

d) deoxygenation of the sulphoxy group employing phosphoruspentachloride in the presence of an olefinic compound having at leastone carbon-carbon double bond with no more than two hydrogen atomsattached thereto, and optionally in the presence of added or alreadypresent catalyst or --CH additive,

e) an imidechloride forming reaction splitting the 7β-acylaminosubstituent according to known procedures by adding sequentiallyN,N-dimethylaniline or other suitable tertiary amine and phosphoruspentachloride,

f) a reaction of the imidechloride with an alcohol such as isobutanol or1,3-dihydroxypropane to form the corresponding iminoether and/or the7-amino-cephem derivative,

f') optional addition of sodium iodide,

f") optional formation of a 7β-(cyclo)alkylideneimino/ammonio derivativeby adding a ketone and

g) introduction of the substituent R₁ or R₂ by replacement of thebromine in the 3-bromomethyl group, introduced in step b), and ifnecessary, water for hydrolysis of the ammonio group;

when A is ##STR11## and P, Q, R₁₋₅ and X are as defined above, and B is##STR12## and T, R₆ and R₇ are as defined above:

by carrying out reactions a-f as described above, and adding a ketone R₄R₅ CO;

when A is --NH₂ or --NH₂.HX, Q is --CH₂ X and P and X are as definedabove, and

B is ##STR13## and T, R₆ and R₇ are as defined above:

by carrying out reactions a-f as described above,

and if necessary, adding water for hydrolysis of the ammoniogroup;

when A is ##STR14## Q is --CH₂ Br and P, X, R₄ and R₅ are as definedabove, and

B is ##STR15## and T, R₆ and R₇ are as defined above:

by carrying out reactions a-f as described above,

and adding a ketone R₄ R₅ CO in the presence of an acid other than HCl;

when A is ##STR16## Q is --CH₂ Cl and P, X, R₄ and R₅ are as definedabove, and

B is ##STR17## and T, R₆ and R₇ are as defined above:

by carrying out reactions a-f as described above,

and adding a ketone R₄ R₅ CO in the presence of hydrogen chloride or inthe presence of a chloride providing agent together with a nitrogencontaining base;

when A is --NH₂ or --NH₂.HX, Q is --CH₂ R₁ or --CH₂ .sup.⊕ R₂ or --CH₂.sup.⊕ R₂ X.sup.⊖ and P, R₁₋₃ and X are as defined above, and

B is ##STR18## and T, R₄ and R₅ are as defined above:

introduction of the substituent R₁ or R₂ by replacement of halogen inthe halomethyl group and, if necessary, water for hydrolysis of theammoniogroup;

when A is ##STR19## Q is --CH₂ X, and P, X, R₄ and R₅ are as definedabove, and

B is ##STR20## another one than being prepared or B is --NH₂.HX, and T,X, R₄ and R₅ are as defined above:

addition of a suitable ketone R₄ R₅ CO, corresponding with B;

when A is --NH₂ or --NH₂.HX, Q is --CH₂ X and P and X are as definedabove, and

B is ##STR21## and T, X, R₄ and R₅ are as defined above:

water for hydrolysis of the ammoniogroup.

For instance, compounds of formula I are prepared and isolated fromcompunds of formula II wherein

Q is --CH₂ R₁,

R₁ is (1-sulfomethyl-tetrazol-5-yl)thio, (1-methyl-tetrazol-5-yl)thio,(1,2,3-triazol-5-yl)thio, (1-carboxymethyl-tetrazol-5-yl)thio,(1-(2-dimethylamino)ethyl-tetrazol-5-yl)-thio,

(5-methyl-1,3,4-thiadiazol-2-yl)thio, and A, P and X are as definedabove, and

B is ##STR22## and T, R₆ and R₇ are as defined above;

A is ##STR23## Q=--CH₂ X where X is bromo or chloro, and P is as definedabove, and B is ##STR24## and T, R₆ and R₇ are as defined above;

A is --NH₂ or --NH₂.HCl, Q is --CH₂ X where X is bromo or chloro or amixture thereof, and P is as defined above, and

B is ##STR25## and T, R₆ and R₇ are as defined above;

Q is --CH₂ R₁ or --CH₂ .sup.⊕ R₂ or --CH₂ .sup.⊕ R₂ X.sup.⊖ where

R₁ is (1,2,3-triazol-5-yl)thio, (1-methyl-tetrazol-5-yl)thio,(5-methyl-1,3,4-thiadiazol-2-yl)thio,(6-hydroxy-2-methyl-5-oxo-1,2,4-triazol-3-yl)thio,(1,3,4-thiadiazol-5-yl)thio,

R₂ is (1-pyridinio), (quinuclidinio-1-ylio) (1-methylpyrrolidin-1-ylio)(cyclopenta[b]pyridin-1-ylio),

and A, P and X are as defined above, and

B is --NH₂, --NH₂.HX or ##STR26## where R₄,R₅ is isopropylidene,cyclopentylidene or cyclohexylidene and T and X are as defined above;

A is ##STR27## where

R₄,R₅ is isopropylidene,

R₄,R₅ is isobutylidene,

R₄,R₅ is cyclopentylidene,

R₄,R₅ is cyclohexylidene,

R₄,R₅ is cycloheptylidene,

and P and X are as defined above, and

B is ##STR28## where R₄,R₅ is isopropylidene or cyclopentylidene,another one than being prepared, or B is --NH₂ or --NH₂.HX, and T and Xare as defined above;

A is ##STR29## Q and P are as defined above, and B is --NH₂, and T is asdefined above;

A is --NH₂, Q and P are as defined above, and

B is ##STR30## and T is as defined above.

The present invention also provides new halo-substituted cephalosporinsof the general formula IC and salts and esters thereof ##STR31## wherein

X is a halogen and R₄ and R₅ are defined as above; and newhalo-substituted cephalosporins of the general formula ID and salts andesters thereof ##STR32## wherein

X is bromo or chloro and m is 0 or 1.

Suitable new intermediates are compounds with formula IC

wherein

R₄,R₅ is isopropylidene,

R₄,R₅ is isobutylidene,

R₄,R₅ is cyclopentylidene,

R₄,R₅ is cyclohexylidene,

R₄,R₅ is cycloheptylidene, and

X is bromo or chloro;

and new intermediates with formula 1D

wherein

HX is HCl and --CH₂ X is --CH₂ Br.

The various aspects of the present invention are depicted in scheme I,wherein m is 0 or 1 and n is 0, 1 or 2.

All the reactions involving these new intermediates are carried out ininert organic solvents at -60° to +150° C., preferably at -40° to +100°C. With lower alkyl or (lower)alkyl in a compound, an alkyl group withup to 8 carbon atoms is meant. With R_(n-n+m) is meant: R_(n), R_(n+1) .. . R_(n+m).

With "halogen or a mixture thereof" and "bromo or chloro or a mixturethereof", respectively, is meant that the compounds with formula I andII may be pure compounds regarding X or a mixture of compounds withdifferent halogen atoms and bromo or chloro atoms, respectively.

According to an aspect of the present invention a process is providedfor the preparation of 7β-amino-3-substitutedmethyl-3-cephem-4-carboxylic acid derivatives of formula I (depictedabove) from 7β-acylamino-3-methyl-3-cephem-4-carboxylic acid 1β-oxidederivatives of formula II (depicted above).

The heterocyclic radical R₃ may be pyridine, pyrimidine, pyridazine,pyrrole, imidazole, pyrazole, isoxazole, isothiazole, thiazole,triazole, oxadiazole, thiadiazole, triazine, thiatriazole and tetrazolelinked by a ring carbon atom to the sulphur atom, whereby optionalsubstituents attached to a ring carbon atom of a heterocyclic ringinclude optionally substituted lower alkyl, cyano, chloro,di(lower)alkylamino, (lower)alkyloxy like methoxy,(lower)alkyloxycarbonyl, di(lower)alkylcarbamoyl, hydroxy, sulfo andcarboxy, while ring nitrogen atoms of the heterocyclic thio group canhave attached thereto an optionally substituted lower alkyl group, andoptional substituents attached to the first or the second carbon atom ofa lower alkyl group attached to a carbon or a nitrogen atom of aheterocyclic ring may include di(lower)alkylamino, chloro, cyano,methoxy, (lower)alkyloxycarbonyl, N,N-dimethylcarbamoyl, hydroxy,carboxy and sulfo.

The positively charged nitrogen group R₂ may be for instance a ##STR33##wherein,

R₈ is alkyl, with up to 8 carbon atoms

R₉ is alkyl, with up to 8 carbon atoms

R₁₀ is alkyl, with up to 8 carbon atoms

whereby R₈, R₉ and R₁₀ are the same or different;

or two of them form cycloalkyl with up to 8 carbon atoms and the thirdone is alkyl, with up to 8 carbon atoms, for instance R₈, R₉ togetherwith the nitrogen atom form a pyrrolidine ring, R₁₀ is methyl;

or R₈, R₉ and R₁₀ form with the nitrogen a heterocyclic ring as a1-pyridinium or quinuclidinium group optionally having substituentsattached to the heterocyclic ring.

It will be clear that the possibility of the introduction of a newsubstituent at the end of a sequence of reactions as described in thisapplication will enlarge the flexibility of a manufacture plant andlower the costs of the products produced therein. Thus, an advantage ofthe present process is the introduction of the substituent in a latephase thereof so that the uniformity of the process will be longermaintained.

A further important advantage of this process is that there are nospecial demands relating to the form wherein the thiol compounds have tobe added to the cephalosporin derivatives. Contrary to the processdescribed in the above-mentioned EPA No. 0137534 or in the EuropeanPatent Specification No. 0045760 surprisingly it does not makedifference whether the thiol compound is added as such, as thiolates(with or without crystal water) or as silyl derivative. The wateroptionally present does not cause any side-reaction.

Generally, yields are similar with the yields obtained according to theprocess described in EPA No. 0137534. Sometimes it may be preferable toadd a ketone to the reaction mixture after having carried out theiminoether forming reaction in order to obtain ammonium compounds.

A preferred group of compounds which can be prepared according to themethod of the invention is a group represented by formula IA ##STR34##wherein R₁ is as defined above.

Another preferred group of compounds which can be prepared according tothe method of the invention is a group represented by formula IB, IB' orIB" ##STR35## wherein R₂ is as defined above and X is bromo or chloro ora mixture thereof.

The invention also relates to new intermediates, viz.7β-(cyclo)alkylideneammonio-3-halomethyl-3-cephem-4-carboxylic acidderivatives of the general formula IC (depicted above) and salts andesters thereof,

wherein

X is halogen, and

R₄, R₅ are as defined above; and to7β-amino/ammonio-3-bromomethyl-3-cephem-4-carboxylic acid and7β-amino/ammonio-3-chloromethyl-3-cephem-4-carboxylic acid derivativesof the general formula ID (depicted above) and salts and esters thereofwherein

X is bromo or chloro, and

m is 0 or 1, and to the isolation of these compounds from the reactionmixture.

A number of new cephalosporin derivatives formed in the course of theabove-described process can be isolated. This concerns in the firstplace a mixture of7β-(cyclo)alkylideneammonio-3-bromomethyl-3-cephem-4-carboxylic acid and7β-(cyclo)alkylideneammonio-3-chloromethyl-3-cephem-4-carboxylic acidderivatives. They can be isolated as a crystalline material after addinga ketone to the reaction mixture, simply by filtration. It is alsopossible to carry out the reaction in such a way that either the 3-bromoor 3-chloromethyl compound are formed. The 3-bromomethyl compound can beformed adding an acid other than hydrogen chloride before orsimultaneously with the ketone. The 3-chloromethyl compound, forinstance can be prepared by adding a sufficient amount of hydrogenchloride. Sofar in the literature only cefalosporanic acid derivativeswith an aldimine substituent at the 7-position have been described forinstance by W. A. Spitzer, T. Goodson, R. J. Smithey and I. G. Wright,J. C. Soc. Chem. Comm., 1338 (1972).

According to an aspect of the invention the proportion of both halogencompounds in7β-(cyclo)-alkylideneammonio-3-halomethyl-3-cephem-4-carboxylic acidderivatives can be influenced. Generally, more of the 3-bromomethylcompound seems to be formed in "acid" medium, while more of thechloromethyl compound is formed in a less "acid" medium. If, forinstance, tetraethylammonium bromide is added to the reaction mixture,more of the chloromethyl compound is formed than without addition ofthis compound in spite of a larger amount of bromide present. Adding achloride providing agent like a pentachloride in the presence ofN,N-dimethylaniline does increase the yield of the chloromethylcompound. Generally, if an acid is added, a higher yield of thebromomethyl compound is obtained except in the case of an excess ofhydrogen chloride. In that case the chloromethyl compound is formed inexcess. These 7-(cyclo)alkylideneammonio-3-bromomethyl derivates arevaluable intermediates for the preparation of many antibiotics, whilethe starting materials can be obtained in an economic way frompenicillins.

Furthermore a mixture of 7β-ammonio-3-bromomethyl-3-cephem-4-carboxylicacid and 7β-ammonio-3-chloromethyl-3-cephem-4-carboxylic acidderivatives or the corresponding amino compounds (indicated throughoutthis text as 3-bromo/chloromethyl) can be isolated. For this type ofcompounds intermolecular substitution could be expected, but this doesnot occur. In Spanish patent ES 461095 (no equivalents found) both the3-bromomethyl and the 3-chloromethyl compound have been mentioned in theexamples as starting compounds in a process to prepare7β-amino-3-substituted methyl-3-cephem-4-carboxylic acid compounds inwhich the 7β-amino group temporarily has been protected with analdehyde. In no way in this patent the source of these unknown andusually highly reactive starting compounds has been described, so itseems justified to ignore the only mention of these compounds in thispatent.

According to another aspect of the invention 7β-amino-3-substitutedmethyl-3-cephem-4-carboxylic acid derivatives of formula I, with R₁ isas defined before, can be prepared directly from these new intermediatesreplacing the halogen atom by introduction of the desired substituentand, if necessary, by hydrolysis of the ammoniogroup. For instance,7β-ammonio-4-carboxy-3[(1-pyridinio)-methyl]-3-cephem dichloride can beprepared in this way from3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide in anearly quantitative yield. In this way very interesting startingmaterials are available which easily can be converted in a simpleone-step synthesis into 7β-amino-3-substitutedmethyl-3-cephem-4-carboxylic acid derivatives. An advantage of a processstarting with7β-(cyclo)alkylideneammonio-3-halomethyl-3-cephem-4-carboxylic acidderivatives, compared to the one described in the European PatentApplication No. 0137534, is that virtually no deacetoxy-cephalosporinderivatives will be found in the end-products. The new intermediates(7β-(cyclo)alkylideneammonio-3-halomethyl-3-cephem-4-carboxylic acidderivatives and 7β-amino/ammonio-3-bromo- orchloromethyl-3-cephem-4-carboxylic acid) can also nearly quantitativelybe converted in each other by addition of a suitable ketone or byhydrolysis of the ammoniogroup.

If during the one-pot process by addition of a ketone a mixture of the3-bromomethyl and the 3-chloromethyl compound of a7β-(cyclo)alkylideneammonio- derivative (indicated as3-bromo/chloromethyl) has been formed, this mixture can be used toprepare the 3-substituted methyl derivatives. The same applies to theisolation of 7β-amino/ammonio-3-bromo/chloromethyl-3-cephem-4-carboxylicacid derivatives from the one-pot process mixture. It is also possibleto prepare the pure 3-halomethyl compound, for instance a pure7β-amino/ammonio-3-bromomethyl-3-cephem-carboxylic acid derivative froma corresponding pure 7β-(cyclo)alkylideneammonio-3-bromomethylderivative.

Both for the 7β-(cyclo)alkylideneammonio- as for the7β-amino/ammonio-3-substituted methyl compound it is possible to convertthe salt forming anion into another one.

The following non-limitative examples illustrate the invention. Thepreparation of the solution containing trimethylsilyl protected startingmaterials is described in the following "Preparation", which precedesthe examples.

Preparation

Conversion of 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid1β-oxide to a mixture containing trimethylsilyl7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate 1β-oxide and aminor amount of trimethylsilyl7β-phenylacetamido-2-bromo-3-bromomethyl-3-cephem-4-carboxylate1β-oxide.

According to the process described in European Patent Application No.0137534, a suspension of 113.0 g (316 mmoles in view of the actualcontent of 97.5% by weight) of7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide, of 11.0g (110 mmoles) of succinimide, and of 0.3 g (1.64 mmoles) of saccharinein 2200 ml of dichloromethane was boiled without reflux in nitrogenatmosphere to remove traces of moisture by boiling off 200 ml ofdichloromethane.

Subsequently 40 ml (188 mmoles) of hexamethyl disilazane were added.Upon stirring and passing over the surface of the reaction mixturenitrogen at a rate of 8 l per hour to remove developing ammonia, boilingwas continued at reflux. After about 3 hours the solution was clear andthe generated gas no longer contained ammonia according to titrationwith acid.

Cooling down the solution to room temperature, 5 g (51 mmoles) ofsulfaminic acid were added and thereafter upon stirring cooled to -4° C.Subsequently the mixture was circulated by pumping from a cooledcontainer through a double faced jacket enveloping a turned onfluorescence tube of 140 W with a peak at 350 nm. Operating undernitrogen atmosphere 40 g (225 mmoles) of N-bromo-succinimide were addedat a temperature of -4°±2° C. After 5 minutes a second portion of 40 gof N-bromo-succinimide was introduced, whereupon irradiation wascontinued for about 65 minutes. The resulting solution weighed 2870 g,and contained according to HPLC-assay 0.070 mmol/g of trimethylsilyl7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate 1β-oxide, whichcorresponds to a direct yield of 63.5%. Besides a relatively muchsmaller amount of trimethylsilyl7β-phenyl-acetamido-2-bromo-3-bromomethyl-3-cephem-4-carboxylate1β-oxide was present. In the subsequent examples this byproduct wasreduced in situ to trimethylsilyl7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate 1β-oxide by theaction of a trisubstituted phosphite, after which the content of thisintermediate was again determined by HPLC-assay.

If such solutions containing the mixture of the monobromide and thedibromide are submitted to selective monodebromination directly aftertheir preparation, the yield of the monobromide after debromination ofthe dibromide may be in the order of 70%. In the examples however,stock-solutions were employed normally so that the total content of themonobromide after debromination usually was in the order of 60% withrespect to the initially used amount of7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide, thisbeing the consequence of storing the stock-solution for several days atabout -25° C.

EXAMPLE I

Preparation of the monosodium salt of7β-amino-3-[[(1-sulfomethyl-tetrazol-5-yl)thio]methyl]-3-cephem-4-carboxylicacid from 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid1β-oxide.

a) In the usual fashion, as described in European Patent Application No.0137534, 2920 g of a solution in dichloromethane containing 173.2 mmolesof trimethylsilyl7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate 1β-oxide and 40mmoles of trimethylsilyl7β-phenylacetamido-2-bromo-3-bromomethyl-3-cephem-4-carboxylate 1β-oxidewas prepared by catalyzed hexamethyldisilazane silylation of 113 g (316mmoles) of 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid1β-oxide followed by light-induced bromination with N-bromo-succinimidein situ.

Continuously applying anhydrous conditions the solution obtained wastreated in situ with 31.4 ml (116 mmoles) of tributyl phosphite during15 minutes at -18°±3° C., followed by cooling to -58° C. 31.6 ml (242mmoles) of cis-cyclooctene, 4.35 ml (34.3 mmoles) ofN,N-dimethylaniline, 6.5 ml (84.3 mmoles) of N,N-dimethylformamide and63.1 g (303 mmoles) of phosphorus pentachloride were added sequentiallyin situ, causing a rise in temperature to -47° C. After 30 minutesstirring at -47° C., 65.1 ml (513.5 mmoles) of N,N-dimethylaniline and63.1 g (303 mmoles) of phosphorus pentachloride were introduced,followed by 70 minutes stirring at -48° C. Subsequently 210 ml (226mmoles) of isobutanol were added at -60° C. followed by 2 hours stirringat about -45° C., resulting in a mixture of7β-ammonio-3-bromomethyl-3-cephem-4-carboxylic acid chloride andcorresponding iminoethers of the 3-bromomethyl and/or 3-chloromethylcompounds.

b) To the well stirred solution obtained as described hereinabove wasadded in 3 minutes a solution of 95 g (90.5%, 358 mmoles) of thedisodium salt of 1-sulfomethyl- tetrazol-5-yl-thiol in 400 ml of water,whereby a temperature of -13° C. was attained. The resulting mixture wasstirred additionally for 22 minutes at -15° C. to -8° C., whereupon 65ml of 25% sodium hydroxide in water were introduced to raise the pH fromabout -0.85 to about -0.15° at 0° C. After standing for 25 minutes at 0°C. the layers were separated and the organic phase was extracted with120 ml of water. After removal by filtration of a small amount ofprecipitate, the aqueous layers combined were washed with 200 ml ofdichloromethane and thereafter treated sequentially with 5 g (26 mmoles)of sodium metabisulphite, 950 ml of methanol and 208.5 ml of 25% sodiumhydroxide in water to give pH 4.05. The resulting solution was leftstanding for about 16 hours at 3° C., whereupon the precipitatedcrystalline product was collected by filtration, followed by washingswith 75 ml of a 3:2 mixture of methanol and water, and with acetone,respectively. After drying in vacuo at 45° C., 75.98 g of a monosodiumsalt of7β-amino-3-[[(1-sulfomethyl-tetrazol-5-yl)thio]methyl]-3-cephem-4-carboxylicacid were obtained in the form of heavy small cream-coloured crystalls.According to HPLC-assay the purity of the isolated product was 82% byweight, from which it is calculated that the actual overall yieldstarting from 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid1β-oxide was 46%. This product could be purified as follows:

It was mixed with 335 ml of water in a 1.5 l beaker glass, followed bydissolution by means of adding 46.7 ml of 4N sodium hydroxide to give pH8. While stirring, 4 g of sodium metabisulphite, 30 g of activatedcarbon and 650 ml of methanol were added, whereupon the pH wasreadjusted to 8 by addition of 2.8 ml of 4N hydrochloric acid. Themixture was filtered through a Seitz filter and the filter cake waswashed with a mixture of 135 ml of methanol and 80 ml of water. Thefiltrates combined were diluted with 325 ml of methanol. While stirring,41.5 ml of 4N hydrochloric acid were introduced slowly to give pH 4.0.The mixture was left standing for 16 hours at 3° C., whereafter theprecipitated crystalline product was washed with 75 ml of 60% methanoland acetone. After drying in vacuo the yield was 64.49 g. As accordingto HPLC assay the purity was 87.5% by weight, the actual overall yieldnow amounted to about 41.5%.

IR (KBr-disc, values in cm⁻¹): 1800, 1615, 1530, 1405, 1340, 1220, 1040,995 and 590.

PMR (D₂ O+NaHCO₃, δ-values in ppm, 60 Mc, int. ref.2,2-dimethylsilapentane-5-sulphonate): 3.21, 3.51, 3.64 and 3.93 (AB-q,J=18 Hz, 2H); 3.95, 4.18, 4.32 and 4.54 (AB-q, J=13.5 Hz, 2H); 4.71 (d,J=4.5 Hz, 1H); 4.99 (d, J=4.5 Hz, 1H); 5.51 (s, 2H).

EXAMPLE II

Preparation of7β-amino-3-[[(1,2,3-triazol-5-yl)thio]methyl]-3-cephem-4-carboxylic acidfrom 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide.

a) According to the method described in Example Ia, 415.4 g of adichloromethane solution containing trimethylsilyl7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate 1β-oxide andtrimethylsilyl7β-phenylacetamido-2-bromo-3-bromomethyl-3-cephem-4-carboxylate 1β-oxide(prepared from 44.73 mmoles of7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide) werefirst treated with 4.44 ml (16.4 mmoles) of tributyl phosphite during 15minutes at -15° C., resulting in a content of 29.43 mmoles oftrimethylsilyl 7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate1β-oxide according to HPLC assay. Thereafter the mixture was submittedin situ to sulphoxide reduction using 5.85 ml (44.9 mmoles) ofcis-cyclooctene, 0.62 ml (8 mmoles) of N,N-dimethylformamide and 9.4 g(45.2 mmoles) of phosphorus pentachloride during 30 minutes at about-45° C., subsequently treated with 9.82 ml (77.5 mmoles) ofN,N-dimethylaniline and 8.4 g (40.2 mmoles) of phosphorus pentachlorideduring 40 minutes at about -45° C., and finally subjected to treatmentwith 30 ml (323 mmoles) of isobutanol during 60 minutes at - 40° C. to-35° C.

b) To the well stirred solution of7β-ammonio-3-bromomethyl-3-cephem-4-carboxylic acid chloride obtained asdescribed hereinabove was added at fast rate a solution of 11.0 g (46.8mmoles) of the pentahydrate of the disodium salt of1,2,3-triazol-5-yl-thiol in 60 ml of water, resulting in a mixture of-10° C. After 60 minutes additional stirring at -10° C., the aqueousphase was separated from the organic phase, which organic phase wasextracted with 20 ml of water. The water-layers combined were firstwashed with 50 ml of dichloromethane, whereupon 0.5 g of sodiummetabisulphite and 125 ml of methanol were added. By slow addition of16.7 ml of 25% sodium hydroxide in water the pH was raised from 0.25 to4.0 at about 10° C. This resulted in an amorphous precipitate, which wascollected by filtration and washed with 25 ml of 60% methanol and withacetone, respectively. After drying in vacuo at 45° C. thecream-coloured7β-amino-3-[[(1,2,3-triazol-5-yl)thio]methyl]-3-cephem-4-carboxylic acidherewith obtained weighed 8.04 g. As according to HPLC assay the purityof the product was 89% by weight, the actual overall yield based on theinitially used amount of7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide was 51%.

IR (KBr-disc, values in cm⁻¹): 1810, 1620, 1540, 1410, 1345, 1290, 1230,1150, 1115, 1060, 1000, 800 and 790.

PMR (DCOOD, δ-values in ppm, 360 Mc, int. ref. TMS): 3.84 and 3.92(AB-q, 2H; J=18 Hz); 4.28 (s, 2H); 5.47 (d, 1H; J=4.5 Hz); 5.50 (d, 1H;J=4.5 Hz); 8.36 (s, 1H).

EXAMPLE III

Preparation of7β-amino-3-[[(1-carboxymethyl-tetrazol-5-yl)thio]methyl]-3-cephem-4-carboxylicacid from 7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylic acid1β-oxide.

a) A process was carried out described in Example Ia. Silylation,bromination and debromination in one pot applied on the startingmaterial 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide(43.2 mmoles) afforded 325 ml of a solution in dichloromethane,containing 26.6 mmoles of trimethylsilyl7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate 1β-oxideaccording to HPLC-assay. Then 5.2 ml (40 mmoles) of cis-cyclooctene,0.71 ml of N,N-dimethylaniline, 1.35 ml of N,N-dimethylformamide, and9.9 g (47.5 mmoles) of phosphorus pentachloride were added at -45° C.,followed by 30 minutes additional stirring at -45° C. 10.6 ml (83.6mmoles) of N,N-dimethylaniline and 9.9 g (47.5 mmoles) of phosphoruspentachloride were further introduced, whereafter the mixture wasstirred again for 30 minutes at the same temperature. Addition of 31.5ml of isobutanol and 60 minutes additional stirring were carried out at-35° C.

b) To the solution of crude7β-ammonio-3-bromomethyl-3-cephem-4-carboxylic acid chloride thusprepared were added 10.6 g (52 mmoles) of the disodium salt of1-carboxymethyl-tetrazol-5-yl-thiol. The temperature of the well stirredmixture was raised to 0° C., followed by 60 minutes stirring at 0° C.After introduction of 50 ml of cold water and 15 minutes stirring at 0°C., the organic phase was separated from the aqueous phase. The organicphase was two times extracted with 40 ml of cold water and wasthereafter discarded. The water-layers combined were washed withdichloromethane, and thereafter diluted with 300 ml of cold methanol.After slow addition of 4N sodium hydroxide to give pH 3.6 thepreparation was left standing overnight at 0° C. The precipitate formedwas collected by filtration, washed with a cold 1:1 mixture of methanoland water and with acetone, respectively, and thereafter dried in vacuoover phosphorus pentaoxide. Isolated were 7.5 g of a crude product whichapart from impurities contained the desired compound for about 75% asthe amino dicarboxylic acid and 25% as a monosodium salt thereof, whichwas revealed by titration with sodium hydroxide on a small sample.According to PMR-assay with the help of a weighed amount of an internalreference the crude product contained 16.53 mmoles of7β-amino-3-[[(1-carboxymethyl-tetrazol-5-yl)thio]methyl]-3-cephem-4-carboxylicacid/-carboxylate (a purity of 87% by weight) corresponding with anoverall actual yield of 38.6%, as calculated from the amount of7β-phenylacetamido-3-methyl-3 -cephem-4-carboxylic acid 1β-oxide.

c) The title compound devoid of remaining monosodium salt was obtainedin about 34% overall yield and with a purity of 95.6% by weightaccording to PMR-assay by dissolution of the crude product in a minimalvolume of water with the help of 4N hydrochloric acid to pH 1.4,dilution with three parts of methanol, filtration and addition of 4Nsodium hydroxide to pH 2.6. The precipitate was collected by filtration,washed with a small volume of a cold 1:1 mixture of methanol and waterand with acetone, and dried in vacuo to constant weight.

IR (KBr-disc, values in cm⁻¹): 1820, 1635, 1550, 1370, 1130, 1080.

PMR (DCO₂ D, δ-values in ppm, 250 Mc, int. ref. TMS): 3.83, 3.90, 3.90,3.97 (AB-q, J=18 Hz, 2H); 4.50, 4.55, 4.64, 4.69 (AB-q, J=14 Hz, 2H);5.43, 5.45, 5.47, 5.49 (AB-q, J about 5 Hz, 2H); 5.50 (s, 2H).

EXAMPLE IV

Preparation of7β-amino-3-[[(1-(2-dimethylamino)ethyl-tetrazol-5-yl)thio]methyl]-3-cephem-4-carboxylicacid from a solution of crude7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylic acid 1β-oxide.

a) A process was carried out as described in Example Ia. Silylation,bromination and debromination in one pot applied on the startingmaterial 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide(33.1 mmoles) afforded 260 ml of a solution in dichloromethane,containing 19.9 mmoles of trimethylsilyl7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate 1β-oxideaccording to HPLC-assay. At -55° C. were added 3.80 ml (29.2 mmoles) ofcis-cyclooctene and 7.66 g (36.8 mmoles) of phosphorus pentachloride,followed by 30 minutes stirring at -45° C. Then 7.62 ml (60.1 mmoles) ofN,N-dimethylaniline and 7.66 g (36.8 mmoles) of phosphorus pentachloridewere introduced at -45° C. After 30 minutes stirring at the sametemperature 27 ml of isobutanol were added at -35° C., whereupon themixture was stirred for 60 minutes at -35° C.

b) To the well stirred solution obtained as described hereinabove wereadded 6.37 g (36.8 mmoles) of pulverized1-(2-dimethylamino)ethyl-tetrazol-5-yl-thiol followed by stirring for 60minutes at -10° C. and for 30 minutes at 0° C. Stirring was continuedduring 10 minutes after dilution with 100 ml of cold water, whereuponthe aqueous phase was separated from the organic phase which wasextracted twice with 50 ml of cold water. The aqueous layers combinedwere washed with dichloromethane, and thereafter stirred with activatedcarbon which was removed by filtration. Triethylamine was added to thefiltrate to give pH 3.2, whereupon the solution was concentratedazeotropically in vacuo with n-butanol to give a volume of about 100 ml.Ethanol was added slowly to achieve complete precipitation. Whilestirring the preparation was kept at 0° C. during 45 minutes, whereuponthe precipitate was collected by filtration, followed by washings with70% ethanol, 96% ethanol and acetone, respectively. After drying invacuo the yield of7β-amino-3-[[(1-(2-dimethylamino)ethyl-tetrazol-5-yl)thio]methyl]-3-cephem-4-carboxylicacid chloride was 5.4 g. As indicated by quantitative PMR-assay on asolution of weighed amounts of the product and of a reference compound,the purity was 88% by weight. Calculating from the initially employedamount of 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid1β-oxide the actual overall yield was 35.2%.

PMR (D₂ O, δ-values in ppm, 60 Mc, int. ref.2,2-dimethylsilapentane-5-sulphonate): 3.07 (s, 6H); 3.77, 3.86, 3.96(t, J=6 Hz, 2H); 3.83 (s, 2H); 4.32 (s, 2H); 4.92, 5.02, 5.12, 5.21 (t,J=6 Hz, 2H); 5.12, 5.21 (d, J=5.2 Hz, 1H); 5.27, 5.35 (d, J=5.2 Hz, 1H).

IR (KBr-disc, values in cm⁻¹): 3360, 1802, 1618, 1530, 1410, 1345, 1285.

EXAMPLE V Preparation of7β-amino-3-[[(1-methyl-tetrazole-5-yl)thio]methyl]-3-cephem-4-carboxylicacid from 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylate 1β-oxide.

In the usual fashion, as described in the European Patent ApplicationNo. 0137534, a suspension of 100 g (purity 97.5%; 280 mmoles) of7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide in 2000ml of dichloromethane was silylated with hexamethyl disilazane andsubsequently brominated with N-bromo-succinimide giving 2855 g of areaction mixture containing a mixture of trimethylsilyl7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate 1β-oxide andtrimethylsilyl7β-phenylacetamido-2-bromo-3-bromomethyl-3-cephem-4-carboxylate1β-oxide. Continuous applying anhydrous conditions 248.8 g of thisstock-solution were cooled down to -60° C. and treated in situ with 2.5ml (9.2 mmoles) of tributyl phosphite for 45 minutes. According to HPLCanalysis, the reaction mixture contained 14.89 mmoles of trimethylsilyl7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate, whichcorresponds to a yield of 61%. 2.7 ml (20.7 mmoles) of cyclooctene, 0.3ml (2.7 mmoles) of N,N-dimethyl aniline and 0.5 ml (6.8 mmoles) ofN,N-dimethylformamide were then added in situ followed by 5 g (24.1mmoles) of phosphorus pentachloride after 5 minutes. After stirring foranother 40 minutes 5.1 ml (40.5 mmoles) of N,N-dimethylaniline and 5 g(24.1 mmoles) of phosphorus pentachloride were added. After stirring for200 minutes at -50° C. a solution of 4.5 g (30 mmoles) of sodium iodidein 100 ml of methanol was added. The temperature was allowed to rise to-10° C. and stirring was continued for 105 minutes. After adding 4.36 g(37.5 mmoles) of 5-mercapto-1-methyl-tetrazole, stirring for 50 minutes,keeping the reaction mixture overnight and stirring for another 180minutes, the reaction mixture was added dropwise to a chilled mixture of130 ml of water, 20 ml of butyl acetate and 8.3 g of sodium bisulphite.Meanwhile the pH was kept at 8 with a 4N potassium hydroxide solution.After separating the layers and extracting the organic layer with 3.5 mlof water, the combined water layers were washed with 25 ml of butylacetate and brought to pH 3.5 with a 4N hydrochloric acid solution.After maintaining the mixture at 2° C. for 16 hours, the precipitate wasfiltered off, washed subsequently with a 3:7 mixture of propanol-2 andwater, a 1:1 mixture of acetone and water and acetone, respectively, anddried in vacuo, thus yielding 3.27 g of the title compound. According toHPLC essay the purity was 83% by weight. The mother liquor containedanother 6% of the title compound. The isolated overall yield startingfrom 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxideamounted to 33%.

EXAMPLE VI

Preparation of7β-amino-3-[[(5-methyl-1,3,4-thiadiazol-2-yl)thio]methyl]-3-cephem-4-carboxylicacid from 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylate 1β-oxide.

341.6 g of the stock solution obtained as described in example V,containing a mixture of trimethylsilyl7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate 1β-oxide andtrimethylsilyl7β-phenylacetamido-2-bromo-3-bromomethyl-3-cephem-4-carboxylate1β-oxide, were treated with 3.4 ml (12.4 mmoles) of tributyl phosphitefor 45 minutes at -60° C. Next, the reduction of the sulphoxide wascarried out with 3.7 ml (28.1 mmoles) of cyclooctene, 0.5 ml (3.7mmoles) of N,N-dimethylaniline, 0.7 ml (9.2 mmoles) ofN,N-dimethylformamide and 6.8 g (32.6 mmoles) of phosphoruspentachloride. After stirring for 40 minutes at -60° C. 7 ml (54.9mmoles) of N,N-dimethylaniline and 6.8 g (32.6 mmoles) of phosphoruspentachloride were added and stirring was continued for 200 minutes at-50° C. After adding a solution of 6.1 g (40.6 mmoles) of sodium iodidein 100 ml of methanol, the temperature was allowed to rise to -10° C.and stirring was continued for 105 minutes. 6.7 g (50.8 mmoles) of2-mercapto-5-methyl-1,3,4-thiadiazole were then added, after which thereaction mixture was stirred for one hour at -10° C. and kept overnightat 0° C. After adding 50 ml of water and stirring for 15 minutes at 0°C., the layers were separated and the organic layer extracted withanother 50 ml of water. To the combined water layers 50 ml of isobutanolwere added and the pH was adjusted to 7.9 with ammonia. After separatingthe layers and extracting the isobutanol layer with water, the pH of thecombined water layers was adjusted to 3.5 with a 4N sulfuric acidsolution. After keeping the reaction mixture for 20 hours in therefrigerator the precipitate was filtered off, washed with water andacetone and dried in vacuo, yielding 5.85 g of the title product. Thepurity according to HPLC was 86.6%. The overall yield amounted to 44%.

EXAMPLE VII

Preparation of7β-amino-[[(1,2,3-triazol-5-yl)thio]methyl]-3-cephem-4-carboxylic acidfrom 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide inthe presence of acetone.

a) According to the method described in Example Ia, 526.5 g of adichloromethane solution containing trimethylsilyl7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate 1β-oxide andtrimethylsilyl7β-phenylacetamido-2-bromo-3-bromomethyl-3-cephem-4-carboxylate 1β-oxidewere cooled down to -52° C. and treated with 5.25 ml (19.4 mmoles) oftributyl phosphite during 48 minutes at -50°±2° C.

Thereafter the mixture was submitted in situ to sulphoxide reductionusing 5.7 ml (43.7 mmoles) of ciscyclooctene, 0.75 ml (5.9 mmoles) ofN,N-dimethylaniline, 1.1 ml (14.3 mmoles) of N,N-dimethylformamide and10.6 g (50.9 mmoles) of phosphorus pentachloride during 30 minutes atabout -45° C., subsequently treated with 10.9 ml (86.0 mmoles) ofN,N-dimethylaniline and 10.6 g (50.9 mmoles) of phosphorus pentachlorideduring 4 hours and 5 minutes at about -50° C., and finally subjected totreatment with 40 ml (431 mmoles) of isobutanol and 225 ml (3.06 mol) ofacetone. The obtained solution was stirred during one hour at -40° C.whereafter it was preserved during the night in the refrigerator at -28°C. The reaction mixture was stirred for another two hours at -25° C.Stirring a sample was taken which contained3-bromo/chloromethyl-4-carboxy-7β-isopropylideneammonio- 3-cephemchloride.

To the well stirred solution obtained as described hereinabove was addedat fast rate a solution of 13.0 g (55.3 mmoles) of the dihydrate of thedisodium salt of 1,2,3-triazol-5-yl-thiol in 70 ml of water, resultingin a mixture of -5° C. Because a separation of the organic and waterlayer could not be obtained the solution was evaporated to a volume of230 ml at the rotavapor at low temperature (0° C.). To the concentrateobtained after about half an hour 100 ml of trichloromethane and 25 mlof H₂ O were added whereafter the layers were separated. Thewater-layers combined were first washed with 10 ml of trichloromethane,whereupon 0.6 g of sodium metabisulphite and 150 ml of methanol wereadded. By slow addition of 25% sodium hydroxide in water under coolingthe pH was raised from 0.3 to 1.7 at about 15° C. Then the pH was raisedwithin 1.5 hour to pH 2.4 at room temperature where crystallizationoccurs. Finally the pH was brought at 3.93 within 10 minutes. There was38 ml of 25% (7.95N) sodium hydroxide used. After a night standing inthe refrigerator the crystal was filtered and washed with 25 ml of 60%methanol and with acetone, respectively. After drying in vacuo at 40° C.the cream-coloured 7β-amino-3-[[(1,2,3-triazol-5-yl)thio]methyl]-3-cephem-4-carboxylic acid herewith obtained weighed 9.0625 g.As according to HPLC assay the purity of the product was 85.5% byweight, the actual overall yield based on the initially used amount of7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide was 48%.The structure was confirmed by spectroscopic means.

EXAMPLE VIII

Preparation of 3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephembromide from a solution of7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide.

a) According to the method described in Example Ia, 390.65 g of adichloromethane solution containing trimethylsilyl7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate 1β-oxide andtrimethylsilyl7β-phenylacetamido-2-bromo-3-bromomethyl-3-cephem-4-carboxylate 1β-oxidewere cooled down to -65° C. and treated with 3.85 ml (14.2 mmoles) oftributyl phosphite during 70 minutes at -60°±2° C.

Thereafter the mixture was submitted in situ to sulphoxide reductionusing 4.2 ml (32.2 mmoles) of ciscyclooctene, 0.5 ml (3.9 mmoles) ofN,N-dimethylaniline, 0.8 ml (10.4 mmoles) of N,N-dimethylformamide and7.65 g (36.7 mmoles) of phosphorus pentachloride during 65 minutes atabout -66° C., subsequently treated with 4.8 ml (37.9 mmoles) ofN,N-dimethylaniline and 7.65 g (36.7 mmoles) of phosphorus pentachlorideduring 2 hours and 15 minutes at about -66° C., and subjected totreatment with 28 ml (30.3 mmoles) of isobutanol. After 1 hour and 30minutes stirring at about -65° C. the reaction mixture was finallysubjected to treatment with 345 ml (4.7 mol) of acetone and 19 ml (166mmoles) of a solution of HBr, 47%.

The crystal was filtered and washed with methylene chloride and withacetone. After drying in vacuo at 40° C. the white-pink coloured3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromideherewith obtained weighed 8.1387 g.

IR-spectrum (KBr-disc; values in cm⁻¹): 3420, 1790, 1692, 1650, 1610,1512, 1397, 1347, 1215, 1180, 1091, 1059, 992, 820, 720, 697, 620.

NMR-spectrum (360 MHz; CF₃ CO₂ D; δ-values in ppm; int. ref. maleicacid; δ=6.35): 2.54 (s, 3H); 2.63 (s, 3H); 3.53, 3.58 (AB-q or d, 2H;J=17.3 Hz); 4.29, 4.33 (AB-q or d, 2H; J=10.7 Hz); 5.33 (d, 1H; J=4.5Hz); 5.83 (d, 1H; J=4.5 Hz).

EXAMPLE IX

Preparation and isolation of 7β-ammonio-3-bromomethyl-4-carboxy-3-cephemchloride from 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid1β-oxide.

According to the process described in Example Ia, 28.91 mmoles of7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide indichloromethane were converted by silylation and bromination into 267.7g of a reaction mixture, containing trimethylsilyl7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate 1β-oxide and aminor amount of its 2-bromo derivative. The solution was in the usualway treated with 3.2 ml of tributyl phosphite to give a solutioncontaining 17.63 mmoles of trimethylsilyl7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate 1β-oxide.

The solution obtained was cooled down to -45° C. followed by theintroduction of 3.86 ml (30 mmoles) of cis-cyclooctene, 0.42 ml (3.3mmoles) of N,N-dimethylaniline, 0.38 ml (5 mmoles) ofN,N-dimethylformamide and 6 g (28.8 mmoles) of phosphorus pentachloride,respectively, whereafter the mixture was stirred for 30 minutes at -45°C.

6 ml (47.6 mmoles) of N,N-dimethylaniline and 6 g (28.8 mmoles) ofphosphorus pentachloride were added at the same temperature. Afteradditional stirring during 30 minutes 22 ml (238 mmoles) of isobutanolwere added slowly, followed by 60 minutes stirring at -35° C.

To the resulting reddish brown solution were added 2.6 ml (31.9 mmoles)of pyridine and 4.1 ml (31.9 mmoles) of trimethylsilyl chloride,followed by 30 minutes stirring at -10° C. The preparation was storedfor 2 hours at -25° C. resulting in the formation of a precipitate. Theprecipitate was collected by filtration and washed with dichloromethane.The product obtained was dissolved in a minimal volume of methanol at 0°C., followed by slow addition of 10 ml of cold 1N hydrochloric acid and200 ml of isopropanol, respectively. The resulting solution wasconcentrated in vacuo to a small volume. The almost white crystallineproduct formed hereby was collected by filtration, sequentially washedwith cold isopropanol and n-hexane. After drying in vacuo the yield was2.64 g. By PMR-assay, using a solution in DCO₂ D prepared from weighedamounts of isolated product and maleic acid, it was found that theisolated product was the hydrochloric salt of7β-amino-3-bromomethyl-3-cephem-4-carboxylic acid in about 85% purity byweight. Therefore, the actual overall yield was about 24%.

PMR (DCO₂ D, δ-values in ppm, 300 Mc, int. ref. TMS): 3.79, 3.85, 3.91(AB-q, J=18 Hz, 2H); 4.68, 4.72, 4.75, 4.79 (AB-q, J=12 Hz, 2H); about5.50 (unsharp q, 2H).

IR (KBr-disc, values in cm⁻¹): 1865, 1630, 1550, 1420, 1360, 1135, 1065,1018.

In zwitter-ionic form 7β-amino-3-bromomethyl-3-cephem-4-carboxylic acidwas also obtained, albeit in much less pure state, from not purified7β-phenylacetamido-3-bromomethyl-4-carboxylic acid 1β-oxide by,sequentially, silylation with trimethylchlorosilane andN,N-dimethylaniline, sulphoxide reduction and imidechloride formationwith phosphorus pentachloride and N,N-dimethylaniline (with or withoutpartial substitution of N,N-dimethylaniline by cis-cyclooctene),treatment with isobutanol and thereafter with a minimal volume of water,and finally isolation of the precipitated compound by addition ofsodiumbicarbonate to pH 3.5.

IR (KBr-disc, values in cm⁻¹): 1860, 1620, 1540, 1410, 1350, 1125, 1060,1015.

EXAMPLE X

Preparation of 3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephemchloride from a solution of7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide undervarious reaction conditions.

a) According to the process described in Example Ia, 287 mmoles of7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide in 2 ldichloromethane were converted into a stock-solution containingtrimethylsilyl 7β-phenylacetamido-3-bromomethyl-3-cephem-4-carboxylate1β-oxide and a minor amount of trimethylsilyl7β-phenylacetamido-2-bromo-3-bromomethyl-3-cephem-4-carboxylate 1β-oxideand brought to -30° C. 5.0 ml of tributyl phosphite was added to afourth part by weight of this stock solution during 20 minutes at -25°C.--30° C. to reduce the dibromide into the monobromide.

The solution obtained hereinabove was cooled down to -60° C., followedby the introduction of 1 ml of N,N-dimethylaniline, 1.5 ml ofN,N-dimethylformamide, 7.5 ml of cis-cyclooctene and 15 g of phosphoruspentachloride, whereupon the mixture was stirred for 30 minutes at -55°C.--60° C.

15 ml of N,N-dimethylaniline and 15 g of phosphorus pentachloride wereadded, followed by 120 minutes stirring at -50° C.--55° C. Subsequently52.5 ml of isobutanol were added, followed by 60 minutes stirring at-25° C.--30° C.

b) The solution of 7β-ammonio-3-bromomethyl-4-carboxy-3-cephem chloridewas divided in seven parts by weight. To each part 90 ml of acetone,together with the additives indicated in the table A, were added and thetemperature was raised to 20° C.-25° C., then cooled down to 5° C. andstored overnight in the refrigerator. The precipitate was collected byfiltration, washed with dichloromethane and acetone and dried in vacuoto constant weight at 20° C.-25° C.

The results have been mentioned in table A. The purity of the isolatedmaterial has been established by quantitative assay being 70%. Theproportion 3-bromomethyl substituted compounds (Br) versus3-chloromethyl substituted compounds (Cl) and the yield of3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem chloride, weregiven in each entry.

                  TABLE A                                                         ______________________________________                                                    proportion                                                        additive      Br:Cl     yield (g)                                                                              yield Br (%)                                 ______________________________________                                        1.     --         70:30     1.91   24.7                                       2.   1 ml H.sub.2 O                                                                             84:16     2.03   31.5                                       3.   5 ml H.sub.2 SO.sub.4, conc                                                                94:06     1.61   27.9                                       4.   5 ml H.sub.3 PO.sub.4, 85%                                                                 85:15     1.85   29.1                                       5.   5 ml HBr, 47%                                                                              94:06     1.96   34.0                                       6.   10 ml HBr, 47%                                                                             94:06     2.03   35.3                                       7.   15 ml HBr, 47%                                                                             93.07     1.85   31.8                                       ______________________________________                                    

EXAMPLE XI

Preparation of3-bromomethyl-4-carboxy-7β-isopropylidene-ammonio-3-cephem chloride froma solution of 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid1β-oxide under various reaction conditions.

Exactly in the same way as described in example X3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem chloride wasprepared except that the additives were introduced 15 minutes before theacetone. The results are given in table B.

                  TABLE B                                                         ______________________________________                                                    proportion                                                        additive      Br:Cl     yield (g)                                                                              yield Br (%)                                 ______________________________________                                        1.     --         49:51     2.33   21.1                                       2.   1 ml H.sub.2 O                                                                             76:24     2.24   31.5                                       3.   5 ml H.sub.2 SO.sub.4, conc                                                                91:09     2.19   36.8                                       4.   5 ml H.sub.3 PO.sub.4, conc                                                                80:20     2.22   32.8                                       5.   5 ml HBr, 47%                                                                              90:10     2.27   37.7                                       6.   10 ml HBr, 47%                                                                             89:11     2.23   36.7                                       7.   15 ml HBr, 47%                                                                             88:12     2.08   33.8                                       ______________________________________                                    

EXAMPLE XII

Preparation of4-carboxy-3-chloromethyl-7β-isopropylidene-ammonio-3-cephem chloridefrom a solution of 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylicacid 1β-oxide under various reaction conditions.

Exactly in the same way as described in example X4-carboxy-3-chloromethyl-7β-isopropylideneammonio-3-cephem chloride wasprepared. The results are given in table C.

                  TABLE C                                                         ______________________________________                                                    proportion                                                        additive      Cl:Br     yield (g)                                                                              yield Cl (%)                                 ______________________________________                                        1.     --         42:58     2.01   17.7                                       2.   2.5 ml DMA*  56:44     1.96   23.0                                       3.   2.5 ml DMA* +                                                                              58:42     2.02   24.6                                            2.5 g PCl.sub.5                                                          4.   2.5 ml PCl.sub.5                                                                           43:57     1.96   17.7                                       5.   15 ml HCl    94:06     1.40   27.6                                       ______________________________________                                         *DMA = N,Ndimethylaniline.                                               

EXAMPLE XIII

Preparation of7β-amino-3-[[(1-methyl-tetrazol-5-yl)thio]methyl]-3-cephem-4-carboxylicacid from 3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephembromide.

800 mg (79% purity; 1.53 mmoles) of3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide wereadded portionwise over about 30 minutes to a stirred solution of 232 mg(2.0 mmoles) of 5-mercapto-1-methyl-tetrazole in 10 ml of a 1:1 mixtureof propanol-2 and water at 25° C. After adjusting the pH to 2.6 with 3.3ml of 1N sodium hydroxide solution, the reaction mixture was stirred foranother 45 minutes. After centrifugation, washing with a 1:1 mixture ofwater and acetone and with acetone, and drying in vacuo at 45° C. 543 mgof the title compound were obtained (purity according to HPLC: 82%;yield 88%).

IR (KBr-disc; values in cm⁻¹): 1800, 1615, 1535, 1410, 1350, 1290, 1275,1255, 1170, 1120, 1060, 1010, 800, 790.

PMR (DCOOD; δ-values in ppm, 360 Mc, int. ref. TMS): 3.92 (s, 2H); 4.18(s, 3H); 4.54 (s, 2H); 5.46 (d, 1H; J=4.5 Hz); 5.48 (d, 1H; J=4.5 HZ).

EXAMPLE XIV

Preparation of7β-amino-3-[[(1,3,4-thiadiazol-2-yl)thio]methyl]-3-cephem-4-carboxylicacid from 3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephembromide

800 mg (79% purity; 1.53 mmoles) of3-bromomethyl-4-carboxy-7β-isopropylidenammonio-3-cephem bromide wereadded portionwise over about two hours to a well stirred solution of 236mg (2.0 mmoles) of 2-mercapto-1,3,4-thiadiazole in 10 ml of a 1:1mixture of propanol-2 and water at room temperature, while the pH waskept at 2.5 with 1N sodium hydroxide solution. After stirring foranother 30 minutes at pH 2.5 the product was isolated by centrifugationand washed with a 1:1 mixture of water and acetone and with acetone,dried in vacuo at 45° C., thus giving 586 mg of the title compound.

IR (KBr-disc; values in cm⁻¹): 1805, 1620, 1545, 1415, 1370, 1350, 1065and 805.

PMR (CF₃ COOD; δ-values in ppm, 360 Mc, int. ref. TMS): 3.60 (s, 2H);4.43 and 4.58 (AB-q, 2H; J=14.0 Hz); 5.13 (s, 2H); 9.92 (s, 1H).

EXAMPLE XV

Preparation of7β-amino-3-[[(1,2,3-triazol-5-yl)thio]methyl]-3-cephem-4-carboxylic acidfrom 3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide.

To a solution of 246 mg (2.0 mmoles) of the sodium salt of1,2,3-triazol-5-yl-thiol in 3 ml of water 7 ml of acetone were added.After adjusting the pH to 1.7 with 0.5 ml of 1N HCl, 800 mg (purity 79%;1.53 mmoles) of3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide wereadded portionwise over about 85 minutes while the pH was kept at 1.8with 1N NaOH. After stirring for another 25 minutes the pH was adjustedto 2.2. After centrifugation, washing with 7.5 ml of a 1:1 mixture ofwater and acetone and with acetone, and drying is vacuo at 45° C., 378mg of the title compound was obtained (purity according to HPLC 78%;overall yield 61.5%). The structure was confirmed by spectroscopicmeans.

EXAMPLE XVI

Preparation of7β-amino-3-[[(5-methyl-1,3,4-thiadiazol-2-yl)thio]methyl]-3-cephem-4-carboxylicacid from 3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephembromide.

800 mg (purity: 79%; 1.53 mmoles) of3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide wereadded to a solution of 265 mg (2.0 mmoles) of2-mercapto-5-methyl-1,3,4-thiadiazole in 15 ml of a 1:1 mixture ofacetone and water at room temperature. The reaction mixture was shakenin an ultrasonic bath for 5 minutes and after one hour for another 5minutes, diluted with a few milliliters of acetone and centrifugated.After washing the precipitate with acetone and diethylether and dryingin vacuo 500 mg of the title compound were obtained with a purity of84%. The overall yield was 80%.

IR (KBr-disc; values in cm⁻¹): 1805, 1620, 1545, 1515, 1410, 1380, 1150,1060, 800 and 790.

PMR (CF₃ COOD; 360 Mc, int. ref. TMS; δ-values in ppm): 2.83 (s, 3H);3.54 and 3.62 (AB-q, 2H; J=16.2 Hz); 4.37 and 4.55 (AB-q, 2H; J=13.8Hz); 5.11 (d, 1H; J=4.5 Hz); 5.14 (d, 1H; J=4.5 Hz).

EXAMPLE XVII

Preparation of7β-amino-3-[[(5-methyl-1,3,4-thiadiazol-2-yl)thio]methyl]-3-cephem-4-carboxylicacid from3-bromo/chloromethyl-4-carboxy-7β-cyclopentylideneammonio-3-cephembromide.

According to the procedure of Example XVI 454 mg of7β-amino-3-[[(2-methyl-1,3,4-thiadiazol-2-yl)thio]methyl]-3-cephem-4-carboxylicacid were obtained by reaction of a mixture of3-bromomethyl-4-carboxy-7β-cyclopentylideneammonio-3-cephem chloride andthe corresponding chloromethyl compound (800 mg); purity: 66% bromocompound, 20% chloro compound; 1.69 mmoles) with 265 mg of2-mercapto-5-methyl-1,3,4-thiadiazole. Purity 83%, yield 65%.

EXAMPLE XVIII

Preparation of7β-amino-3-[[(5-methyl-1,3,4-thiadiazol-2-yl)thio]methyl]-3-cephem-4-carboxylicacid from 7β-amino-3-bromomethyl-3-cephem-4-carboxylic acid.

A solution of 1N-sodiumhydroxide (about 3.4 ml) was added to 60 mg (0.45mmol) of 2-mercapto-5-methyl-1,3,4-thiadiazole in 2 ml water till thesolution was clear at a pH of 7.5. 100 mg (0.34 mmol) of7β-amino-3-bromomethyl-3-cephem-4-carboxylic acid were addedportionwise. About 2.8 ml of a solution of 0.1N NaOH was necessary tomaintain the pH at about 7.5. Then the pH of the solution was adjustedto 4 with 3.6 ml of a solution of 0.1N HCl. The precipitate formed wasfiltered and washed with water and acetone. After drying in vacuo 61 mgof7β-amino-3-[[(5-methyl-1,3,4-thiadiazol-2-yl)thio]methyl]-3-cephem-4-carboxylicacid were obtained with a purity of 62.5%. The overall yield was 32.5%.

IR (KBr-disc, values in cm⁻¹): about 3410, 1800, 1620, about 1540, 1412,1350, 1060.

EXAMPLE XIX

Preparation of7β-amino-3-[[(6-hydroxy-2-methyl-5-oxo-1,2,4-triazol-3-yl)thio]methyl]-3-cephem-4-carboxylicacid from 3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephembromide.

800 mg (79% purity; 1.53 mmoles) of3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide wereadded portionwise over 80 minutes to a stirred solution of 318 mg (2.0mmoles) of 6-hydroxy-1-mercapto-2-methyl-5-oxo-1,2,4-triazole in 10 mlof a 1:1 mixture of acetone and water at room temperature, while the pHwas kept at 1.95 with a IN NaOH solution (3.2 ml). After stirring foranother 15 minutes the crystalline product was separated bycentrifugation, washed with a mixture of acetone and water, with acetoneand dried in vacuo, thus yielding 626 mg of the title compound. Purity92%; yield 100%.

IR (KBr-disc; values in cm⁻¹): 1795, 1640, 1615, 1585, 1410, 1345, 1290,1220, 1100, 800 and 785.

PMR (D₂ O+NaHCO₃ ; 360 Mc, int. ref. TMS; δ-values in ppm): 3.69 (s,3H); 3.49 and 3.71 (AB-q, 2H; J=18 Hz); 4.09 and 4.40 (AB-q, 2H; J=13.3Hz); 4.80 (d, 1H; J=5 Hz); 5.10 (d, 1H; J=5 Hz).

EXAMPLE XX

Preparation of7β-amino-3-[[(6-hydroxy-2-methyl-5-oxo-1,2,4-triazol-3-yl)thio]methyl]-3-cephem-4-carboxylicacid from3-bromo/chloromethyl-4-carboxy-7β-cyclohexylideneammonio-3-cephemchloride.

According to the procedure of Example XVI 703 mg of7β-amino-3-[[(6-hydroxy-2-methyl-5-oxo-1,2,4-triazol-3-yl)thio]methyl]-3-cephem-4-carboxylicacid were obtained starting from a mixture of3-bromomethyl-4-carboxy-7β-cyclohexylideneammonio-3-cephem chloride andthe corresponding 3-chloromethyl compound (1.3 g; purity: 69% bromocompound, 8.4% chloro compound; 2.76 mmoles) and6-hydroxy-1-mercapto-2-methyl-5-oxo-1,2,4-triazole in 16 ml of a 1:1mixture of acetone and water. The reaction temperature was 40° C. Thepurity of the final product was 92.5%. The yield was 64%.

EXAMPLE XXI

Prepartion of 7β-ammonio-4-carboxy-3-[(1-pyridinio)-methyl]-3-cephemdichloride from3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide.

A mixture of 4.63 g (purity 85%; 9.5 mmoles) of3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide, 50 mlof acetonitrile, 50 ml of acetone and 10 ml (40 mmoles) ofN,O-bis(trimethylsilyl)acetamide was stirred under nitrogen at roomtemperature for 15 minutes and for 30 minutes at 0° C. After adding 10ml (124 mmoles) of pyridine and stirring for 3 hours at 0° C. a clearorange solution was obtained. After pouring out the reaction mixtureinto a mixture of 5 ml of water, 10 ml of isopropanol and 5 ml oftoluene, the solvents were evaporated. The residual solid was dissolvedin 35 ml of a 1N hydrochloric acid solution at 0° C. After adding slowly195 ml of isopropanol and stirring overnight at 0° C., the precipitateformed was centrifugated, washed with isopropanol and diethylether anddried in vacuo, yielding 3.55 g of the title compound. The yield was92%.

IR (KBr-disc; values in cm⁻¹): 1795, 1610, 1487, 1395, 1335, 1280, 1240,1150, 1050, 800, 770 and 680.

PMR (360 MHz; D₂ O; δ-values in ppm): 3.40 and 3.77 (2xd, 2H; J=18 Hz);5.26 (d, 1H; J=4.8 Hz); 5.35 (d, 1H; J=4.8 Hz); 5.45 and 5.64 (2xd, 2H;J=14.4 Hz); 8.16 (t, 2H; J about 6 Hz); 8.64 (t, 1H; J about 6 Hz); 9.00(d, 2H; J about 6 Hz).

EXAMPLE XXII

Preparation of 7β-ammonio-4-carboxy-3-[(1-pyridinio)methyl]-3-cephemdichloride from3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide.

A mixture of 463 mg (purity 85%, 0.95 mmole) of3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide, 5 mlof acetone, 5 ml of acetonitrile and 1.1 ml (4 mmoles) ofN,O-bis(trimethylsilyl)trifluoroacetamide was stirred under nitrogen for30 minutes at 0° C. After addition of 1 ml of pyridine and stirring forthree and a half hour the conversion into the title product wasquantitative according to HPLC-analysis.

2 ml of water, 2 ml of isopropanol and 2 ml of toluene was added, themixture was concentrated in vacuo to dryness and the remaining residuein 3.5 ml of a 1N hydrochloric acid solution. After adding slowly 20 mlof isopropanol and stirring for one and a half hour the mixture wascentrifugated. Washing the precipitate with isopropanol and ether anddrying in vacuo gave 364 mg of the title product with a purity of 93%.The yield was 98%.

EXAMPLE XXIII

Preparation of7β-ammonio-4-carboxy-3-[(1-methylpyrrolidin-1-ylio)-methyl]-3-cephemdichloride from3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide.

463 mg (purity 85%, 0.95 mmole) of3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide wassilylated in a mixture of 5 ml of acetone and 5 ml of acetonitrile with2 ml (8 mmoles) of N,O-bis(trimethylsilyl)-acetamide under nitrogen atroom temperature for 15 minutes. After cooling down and stirring foranother 30 minutes a 0° C., a mixture of 0.24 ml (2.3 mmoles) ofN-methylpyrrolidine, 20 ml of acetone and 20 ml of acetonitrile wasadded dropwise over a period of 60 minutes and stirring was continued at0° C.

After addition of 1.5 ml of a 1N hydrochloric acid solution, 5 ml ofisopropanol and 5 ml of toluene, the reaction mixture was concentratedin vacuo. Then 2.5 ml of a 1N hydrochloric acid solution and 30 ml ofisopropanol was added and the mixture was concentrated to a volume of 20ml and stored at 0° C. The crystalline precipitate formed was obtainedby centrifugation. Washing and drying in vacuo gave 313 mg of the titlecompound with a purity of 72%. The yield was 64%.

IR (KBr-disc, values in cm⁻¹): 1790, 1640, 1620, 1600, 1400, 1345, 1290,1145, 1065, 925 and 800.

PMR (360 MHz; D₂ O; δ-values in ppm): 2.25 (m, 4 Hz); 3.01 (s, 3H); 3.57(m, 4 Hz); 3.62 and 3.98 (2xd, 2H; J=18 Hz); 4.11 and 4.75 (2xd, 2H;J=13.2 Hz); 5.23 (d, 1H; J=4.5 Hz); 5.44 (d, 1H; J=4.5 Hz).

EXAMPLE XXIV

Preparation of7β-ammonio-4-carboxylate-3-[(cyclopenta[b]pyridin-1-ylio)methyl]-3-cephembromide from 3-bromomethyl-4-carboxy-7β-isohexylideneammonio-3-cephembromide.

A mixture of 510 mg (purity 85%, 0.95 mmole) of3-bromomethyl-4-carboxy-7β-cyclohexylideneammonio-3-cephem bromide, 5 mlof methylene chloride and 2 ml (8 mmoles) ofN,O-bis(trimethylsilyl)acetamide was stirred for 15 minutes at roomtemperature and for 30 minutes at 0° C. Then with continuous stirring0.28 ml of 2,3-cyclopenta[b]pyridine in 5 ml of methylene chloride wasadded during 10 minutes at 0° C. and stirring was continued for another3 hours, the temperature for the last 30 minutes being 20° C. Afterconcentrating the reaction mixture in vacuo and dissolving the residuein a mixture of 1 ml of water, 0.3 ml of methanol and 0.5 ml of a 4Nhydrobromic acid solution, 12 ml of isopropanol was added slowly. Afterstanding overnight 370 mg of the title compound was obtained bycentrifugation, washing with acetone and drying.

IR (KBr-disc, values in cm⁻¹): 1805, 1785, 1645, 1620, 1595, 1472, 1417,1350, 1295, 1285, 1146, 1058, 820 and 800.

PMR (360 MHz; D₂ O; δ-values in ppm): 2.18 (m, 2H); 3.11 (t, 2H; J about7.5 Hz); 3.21 (t, 2H; J about 7.5 Hz); 3.47 and 3.65 (2xd, 2H; J=18 Hz);5.23 (s, 2H); 5.58 and 5.65 (2xd, 2H; J=13.2 Hz); 7.92 (t, 1H); 8.43 (d,1H; J about 9 Hz).

EXAMPLE XXV

Preparation of7β-ammonio-4-carboxylate-3-[(quinuclidin-1-ylio)-methyl]-3-cephembromide from 3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephembromide.

A mixture of 525 mg (purity 85%, 0.95 mmole) of3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide, 2 ml(8 mmoles) of N,O-bis(trimethylsilyl)acetamide and 10 ml of1,1,2-trichlorotrifluoroethane was stirred under nitrogen for 45 minutesat 0° C. With continuous stirring and maintaining the temperature at 0°C. 266 mg (2.4 mmoles) of quinuclidine in 10 ml of1,1,2-trichlorotrifluoroethane was added over a period of 2 hours. Afterstirring for another hour 5 ml of isopropanol, 1 ml of methanol and 4 mlof a 1N hydrochloric acid solution was added with vigorous stirring andfurthermore a small amount of water until a clear solution was obtained.After separation of the layers and extracting the organic layer with 2ml of water the combined water layers were concentrated in vacuo untilthe volume of the reaction mixture was about 2 ml. Adding slowly 12 mlof isopropanol, filtering off the crystalline precipitate formed,washing and drying yielded 384 mg of the title product with a purity of89%. The yield was 87%.

IR (KBr-disc, values in cm⁻¹): 3120, 2940, 2885, 2130, 1790, 1620, 1600,1492, 1466, 1415, 1400, 1390, 1380, 1345, 1290, 1195, 1142, 1075, 935,792 and 452.

PMR (360 MHz; D₂ O; δ-values in ppm, determined in the presence ofmaleic acid): 1.7 (m, 6H); 1.9 (m, 1H); 3.2-3.4 (m, 6H); 3.49 and 3.61(2xd, 2H; J about 17 Hz); 3.95 and 4.40 (2xd, 2H; J about 14 Hz); 4.98(d, 1H; J=4.5 Hz); 5.23 (d, 1H; J=4.5 Hz).

EXAMPLE XXVI

Preparation of3-bromo/chloromethyl-4-carboxy-7β-iso-butylideneammonio-3-cephemchloride from 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid1β-oxide.

After treating 373 g of a solution obtained as described in example Iacontaining a mixture of bromo cephem silyl esters with 3.75 ml (13.9mmoles) of tributyl phosphite at -37° C. for one hour the reduction ofthe sulphoxide was carried out with 4.1 ml (31.4 mmoles) of cyclooctene,0.5 ml (3.9 mmoles) of N,N-dimethylaniline, 0.8 ml (10.4 mmoles) ofN,N-dimethylformamide and 7.5 g (36.0 mmoles) of phosphoruspentachloride at -50° C. for 50 minutes. After adding 7.7 ml (60.7mmoles) of N,N-dimethylaniline and 7.5 g (36.0 mmoles) of phosphoruspentachloride and stirring for 195 minutes at -50° C. a mixture of 30 ml(0.32 mmol) of isobutanol and 125 ml (1.39 mmol) of ethyl methyl ketonewas added. Stirring was continued for 30 minutes without cooling and thereaction mixture was kept at 0° C. for 72 hours. Thus the precipitateformed was filtered off and dried in vacuo yielding 7.48 g of a mixtureof the title compounds. The ratio of the bromo compound to the chlorocompound was 2:1. The overall yield starting from7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide amountedto 33%.

PMR-spectrum of the bromomethyl compound (360 MHz; CF₃ CO₂ D; δ-valuesin ppm): 1.22 (t, 3H); 2.53 (s, 3H); 2.89 (m, 2H); 4.28 and 4.34 (2xd,2H; J=10.8 Hz); 5.34 (d, 1H; J=4.5 Hz); 5.81 (d, 1H; J=4.5 Hz).

PMR-spectrum of the chloromethyl compound (ibidem): 1.22 (t, 3H); 2.53(s, 3H); 2.89 (m, 2H); 4.42 (s, 2H); 5.34 (d, 1H; J=4.5 Hz); 5.83 (d,1H; J=4.5 Hz).

EXAMPLE XXVII

Preparation of3-bromo/chloromethyl-4-carboxy-7β-cyclo-pentylideneammonio-3-cephemchloride from 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid1β-oxide.

706 g of a stock-solution obtained as described in example Ia containinga mixture of bromo cephem silyl esters were treated with 7.1 ml (26.2mmoles) of tributyl phosphite and subsequently with 7.7 ml (59.1 mmoles)of cyclooctene, 1 ml (7.9 mmoles) of N,N-dimethylaniline, 1.5 ml (19.5mmoles) of N,N-dimethylformamide and 14.3 g (68.7 mmoles) of phosphoruspentachloride in the usual way. Then 14.65 ml (115.5 mmoles) ofN,N-dimethylformamide and 14.3 g of phosphorus pentachloride were addedand stirring was continued at -50° C. for 210 minutes at -50° C. Thenthe reaction mixture was cooled down to -70° C. and a mixture of 55 ml(592 mmoles) of isobutanol and 25 ml (282 mmoles) of cyclopentanone wasadded. After stirring for 30 minutes at -40° C. followed by standing for30 hours at -25° C. another 100 ml (1.13 mmol) of cyclopentanone wereadded and the temperature was allowed to rise to 28° C. After a further4 hours at 2° C. the resultant precipitate was collected, washed withcyclopentanone and diethyl ether, and dried in vacuo to give 14.4 g of amixture of the title compounds. The overall yield starting from7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide amountedto 45%. The ratio of the bromo compound to the chloro compound was 3:1.

PMR-spectrum of the bromomethyl compound (360 MHz; CF₃ COOD; δ-values inppm): 1.96 (m, 4H); 2.95 (m, 4H); 4.30 (s, 2H); 5.30 (d, 1H; J=4.5 Hz);5.70 (d, 1H; J=4.5 Hz).

PMR-spectrum (ibidem) of the chloromethyl compound: 1.96 (m, 4H); 2.95(m, 4H); 4.39 (s, 2H); 5.30 (d, 1H; J=4.5 Hz); 5.72 (d, 2H; J=4.5 Hz).

EXAMPLE XXVIII

Preparation of3-bromo/chloromethyl-4-carboxy-7β-cyclohexylideneammonio-3-cephemchloride from 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid1β-oxide.

455 g of a stock solution obtained as previously described in example Iacontaining a mixture of bromo cephem silyl esters were treated with 4.4ml (16.3 mmoles) of tributyl phosphite at -50° C. for 45 minutes. Afteradding 4.75 ml (36.4 mmoles) of cyclooctene, 0.65 ml (5.1 mmoles) ofN,N-dimethylaniline, 0.9 ml (11.7 mmoles) of N,N-dimethylformamide and8.8 g (42.3 mmoles) of phosphorus pentachloride stirring was continuedat -50° C. for 45 minutes. Then 9.1 ml (71.8 mmoles) ofN,N-dimethylaniline and 8.85 g (42.5 mmoles) of phosphorus pentachloridewere added, the reaction mixture was stirred at -45° C. for 2 hours anda mixture of 34 ml of isobutanol and 20 ml (193 mmoles) of cyclohexanonewas added, carefully maintaining the temperature below -50° C. Afterstirring for another 90 minutes at -50° C. the reaction mixture was keptovernight at 2° C. The resultant precipitate was filtered off, washedwith acetonitrile and diethylether and dried in vacuo to give 11 g of amixture of the title compounds. The ratio of the bromo compound to thechloro compound was 7:1. The overall yield starting from7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide amountedto 48%.

PMR-spectrum (360 MHz; CF₃ COOD; δ-values in ppm) of the bromomethylcompound: 1.64 (m, 2H); 1.93 (m, 4H); 2.79 (m, 4H); 4.27 and 4.34 (2xd,2H; J=10.5 Hz); 5.34 (d, 1H; J=4.5 Hz); 5.88 (d, 1H; J=4.5 Hz).

PMR-spectrum (ibidem) of the chloromethyl compound: 1.64 (m, 2H); 1.93(m, 4H); 2.79 (m, 4H); 4.39 and 4.45 (2xd, 2H; J=12.6 Hz); 5.34 (d, 1H;J=4.5 Hz); 5.91 (d, 1H; J=4.5 Hz).

EXAMPLE XXIX

Preparation of3-bromo/chloromethyl-4-carboxy-7β-cyclohexylideneammonio-3-cephembromide from 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid1β-oxide.

399 g of a stock solution obtained as previously described in example Iacontaining a mixture of bromo cephem silyl esters were treated with 4.0ml (14.8 mmoles) of tributyl phosphite at -50° C. for 50 minutes. Afteradding 4.3 ml (33.0 mmoles) of cyclooctene, 0.5 ml (3.9 mmoles) ofN,N-dimethylaniline, 0.8 ml (10.4 mmoles) of N,N-dimethylformamide and7.9 g (37.9 mmoles) of phosphorus pentachloride stirring was continuedat about -47° C. for 40 minutes. Then 4.9 ml (38.7 mmoles) ofN,N-dimethylaniline and 7.9 g (37.9 mmoles) of phosphorus pentachloridewere added, the reaction mixture was stirred at -50° C. for 1 hour and35 minutes and 60 ml (650 mmoles) of isobutanol was added. Thetemperature rised to -35° C. for just a moment, then the mixture wascooled again to -50° C. Then 20 ml of a solution of hydrogen bromide,47% (174 mmoles HBr) and thereafter 50 ml (482 mmoles) of cyclohexanonewere added. After stirring for another 2 hours at -50° C. the reactionmixture was kept for 6 days at 2° C. The resultant precipitate wasfiltered off, washed with cyclohexanone and acetone and dried in vacuoto give 9.9 g of a mixture of the title compounds. The ratio of thebromo compound to the chloro compound was 9:1. The overall yieldstarting from 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid1β-oxide amounted to 46%.

IR-spectrum (KBr-disc; values in cm⁻¹): 1798, 1702, 1658, 1621, 1515,1406, 1350, 1211, 1194, 1093, 1060, 996, 987, 702, 620.

NMR-spectrum (360 MHz; CF₃ CO₂ D; δ-values in ppm; int. ref. maleicacid; δ=6.35): 1.5-2.1 (m, 6H); 2.6-2.9 (m, 4H); 3.56 (s, 2H); 4.27,4.33 (AB-q or d, 2H; J=10.5 Hz); 5.32 (d, 1H; J=4.5 Hz); 5.86 (d, 1H;J=4.5 Hz).

EXAMPLE XXX

Influence of the addition of a quaternary ammonium compound on thechloro/bromo ratio of the preparation of4-carboxy-3-chloro/bromomethyl-7β-cyclohexylideneammonio-3-cephemchloride from 7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid1β-oxide.

434.4 g of a stock solution obtained as previously described containinga mixture of bromo cephem silyl esters were treated with 4.2 ml (15.5mmoles) of tributyl phosphite at -55° C. for 1 hour. The reduction ofthe sulphoxide moiety was carried out with 4.55 ml (34.9 mmoles) ofcyclooctene, 0.6 ml (4.7 mmoles) of N,N-dimethylaniline, 0.9 ml (11.7mmoles) of N,N-dimethylformamide and 8.4 g (40.3 mmoles) of phosphoruspentachloride in the usual way. After cooling the reaction mixture downto -66° C., stirring was continued and a mixture of 20 g (95.2 mmoles)of tetraethyl ammonium bromide, 32 ml of isobutanol, 25 ml of methylenechloride and 20 ml (193 mmoles) of cyclohexanone was added. After thereaction mixture was warmed up to 0° C. over 70 minutes, it wasmaintained at 2° C. overnight. The resultant precipitate was filteredoff, washed with methylene chloride and dried to give 9.76 g of amixture of the title compounds. The ratio of the bromo compound to thechloro compound was 2:3. The overall yield starting from7β-phenylacetamido-3-methyl-3-cephem-4-carboxylic acid 1β-oxide amountedto 48%.

EXAMPLE XXXI

Preparation of 7β-amino-3-bromomethyl-3-cephem-4-carboxylic acid from3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide.

In a centrifuge-tube, placed in an ultrasonic bath, 1000 mg (purity 79%,90% bromo compound, 1.91 mmol) of3-bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide weredissolved in 15 ml 2-methoxy ethanol. After adding 1.0 ml of a 2Nhydrogen bromide solution this mixture was shaken and stirred during10-15 minutes and the solution became clear. Stirring and shaking asolution of 0.60 ml (4.73 mmoles) of N,N-dimethylaniline in 15 mlacetonitrile was dropped to the mixture.

The precipitate was centrifugated and washed with 25 ml 80%acetonitrile, acetonitrile and ether, respectively. After drying in anitrogen stream and then in vacuo the yield of7β-amino-3-bromomethyl-3-cephem-4-carboxylic acid was 647.6 mg with apurity of 82.7%. The overall yield was 95.5%.

IR (KBr-disc, values in cm⁻¹): 3445, 3180, 1800, 1616, about 1535, 1410,1350, 1290, 1210, 1150, 1114, 1059, 1001, 800, 791, 519, 432.

EXAMPLE XXXII

Preparation of 7β-amino-3-chloromethyl-3-cephem-4-carboxylic acid from4-carboxy-3-chloromethyl-7β-isopropylideneammonio-3-cephem chloride.

In a centrifuge-tube, placed in an ultrasonic bath, 400 mg (purity93.9%, 1.15 mmol) of7β-isopropylideneammonio-4-carboxy-3-chloromethyl-3-cephem chloride weredissolved in 10 ml 2-methoxyethanol. After adding 1.5 ml of 2N hydrogenchloride and shaking and trilling during about 25 minutes a clearsolution was obtained. During half an hour 0.45 ml (3.55 mmoles) ofN,N-dimethylaniline dissolved in 15 ml acetonitrile was dropped to themixture.

The precipitate was centrifugated and washed with 25 ml 80%acetonitrile, acetonitrile and ether, respectively. After drying in anitrogen stream and then in vacuo the yield of7β-amino-3-chloromethyl-3-cephem-4-carboxylic acid was 207.7 mg with apurity of 95.3%. The overall yield was 68.9%.

IR (KBr-disc, values in cm⁻¹): about 3440, 3180, 1804, 1622, about 1540,1412, 1351, 1293, 1257, 1123, 1061, 1010, 889, 802, 792, 521, 430.

PMR (CF₃ COOD, o-values in ppm, 360 Mc, int. ref. TMS): 3.52, 4.40, 4.43(2H; 2xd, J=11.9 Hz), 5.13, 5.15 (2H; 2xd, J=4.5 Hz).

EXAMPLE XXXIII

Preparation of 7β-ammonio-3-chloro/bromomethyl-3-cephem-4-carboxylatefrom 3-bromo/chloromethyl-4-carboxy-7β-isopropylideneammonio-3-cephemchloride.

A solution of 5 g (9.49 mmoles) of3-chloro/bromomethyl-4-carboxy-7β-isopropylideneammonio-3-cephemchloride (bromomethyl:chloromethyl=45:26) in 75 ml 2-methoxyethanol and25 ml of dry acetone was treated with charcoal. After washing with 25 mlof acetone, slowly 30 ml of a mixture of 11.8 mmoles ofN,N-dimethylaniline in acetone were dropped into the filtrate. Afterstirring for ten minutes the precipitate was filtered and washed withacetone and ether respectively. The yield after drying in vacuo was 2.16g of 7β-ammonio-3-chloro/bromomethyl-3-cephem-4-carboxylate (57.0%3-chloromethyl, 20.7% 3-bromomethyl as was found by spectroscopicmeans). The overall yield was 70%.

IR (KBr-disc, values in cm⁻¹): about 3420, 1800, 1621, about 1545, 1410,1348, 1056, 1006, 799, 790.

EXAMPLE XXXIV

Preparation of3-bromo/chloromethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromidefrom 7β-ammonio-3-bromo/chloromethyl-3-cephem-4-carboxylate.

6.585 g 7β-ammonio-3-bromo/chloromethyl-3-cephem-4-carboxylate(bromomethyl:chloromethyl=31:69) were dissolved in 50 ml concentratedhydrogen bromide and 20 ml water. This mixture was stirred for fiveminutes whereafter 100 ml acetone was slowly added during half an hour.After stirring for one hour, 650 ml of acetone were added.

After four days standing in the refrigerator the precipitate wasfiltered and washed with acetone and ether. The yield after drying invacuo was 8.032 g of3-bromo/chloromethyl-4-carboxy-7β-isopropylideneammonio-3-cephem bromide(36% 3-bromomethyl, 64% 3-chloromethyl as was found by spectroscopicmeans).

EXAMPLE XXXV

Preparation of3-chloromethyl-4-carboxy-7β-cyclopentylideneammonio-3-cephem chloridefrom 7β-ammonio-3-bromo/chloromethyl-3-cephem-4-carboxylate.

In a centrifuge-tube a small amount of7β-ammonio-3-bromo/chloromethyl-3-cephem-4-carboxylate was dissolved ina small amount of a solution of concentrated hydrogen chloride. Anexcess of cyclopentanone was added to the solution.

After standing for a week-end in the refrigerator the product was washedwith cyclopentanone and ether, respectively, filtered off and dried invacuo giving3-chloromethyl-4-carboxy-7β-cyclopentylideneammonio-3-cephem chloride asconfirmed by NMR.

IR (KBr-disc, values in cm⁻¹): about 3410, 1795, 1706, 1681, 1633, 1506,1405, 1353, 1098, 1061, 791, 691.

EXAMPLE XXXVI

Preparation ofchloro/bromomethyl-4-carboxy-7β-cyclohexylideneammonio-3-cephem chloridefrom 7β-ammonio-3-chloro/bromomethyl-3-cephem-4-carboxylate.

200 mg of 7β-ammonio-3-chloro/bromomethyl-3-cephem-4-carboxylate(prepared in example XXXII) were dissolved in 1 ml of a solution ofconcentrated hydrogen chloride. Then 0.5 ml of cyclohexanone was added.After a short time a crystalline precipitate was formed. After stirringfor 20 minutes cyclohexanone (1 ml) was added again, whereafter therewas stirred for another 20 minutes. Then 4 ml of cyclohexanone was addedslowly. After stirring for 10 minutes the product was filtered andwashed with cyclohexanone and ether, respectively.

After drying in vacuo the yield was 235.2 mg of3-chloro/bromomethyl-4-carboxy-7β-cyclohexylideneammonio-3-cephemchloride (80% 3-chloromethyl, 20% 3-bromomethyl as was found with NMR).

IR (KBr-disc, values in cm⁻¹): about 3410, 1800, 1706, 1664, 1630, 1526,1410, 1351, 1187, 1098, 1061, 706, 692.

EXAMPLE XXXVII

Preparation ofchloro/bromomethyl-4-carboxy-7β-cycloheptylideneammonio-3-cephemchloride from 7β-ammonio-3-chloro/bromomethyl-3-cephem-4-carboxylate.

In a centrifuge-tube a small amount of7β-ammonio-3-chloro/bromomethyl-3-cephem-4-carboxylate (prepared inexample XXXIII) was dissolved in a small amount of a solution ofconcentrated hydrogen chloride. To the solution thus obtained an excessof cycloheptanone was added. After about one hour a very fineprecipitate was formed. Warming the mixture and cooling down again, themixture was made somewhat more granulated. The precipitate wascentrifugated and washed with cycloheptanone and twice with ether,respectively.

After drying in vacuo crystalline3-chloro/bromomethyl-4-carboxy-7β-cycloheptylideneammonio-3-cephemchloride (93% 3-chloromethyl, 7% 3-bromomethyl as was found with NMR)was formed.

IR (KBr-disc, values in cm⁻¹): about 3410, 1798, 1710, 1643, 1522, 1405,1358, 1098, 1061, 685.

EXAMPLE XXXVIII

Preparation of3-bromo/chloromethyl-4-carboxy-7β-isobutylideneammonio-3-cephem chloridefrom 3-bromo/chloromethyl-4-carboxy-7β-isopropylideneammonio-3-cephemchloride.

In a centrifuge-tube a small amount of3-bromo/-chloromethyl-4-carboxy-7β-isopropylideneammonio-3-cephemchloride was dissolved in a small amount of concentrated hydrogenchloride. An excess of ethyl methyl ketone was added. No precipitate wasformed.

After standing for a week-end in the refrigerator the formed, whiteprecipitate was filtered and washed with ethyl methyl ketone and ether,respectively. After drying crystalline3-bromo/chloromethyl-4-carboxy-7β-sec-butylideneammonio-3-cephemchloride was obtained.

IR (KBr-disc, values in cm⁻¹): about 3410, 1801, 1708, 1656, 1629, 1516,1405, 1348, 1232, 1183, 1095, 1062, 1000, 708, 689.

EXAMPLE XXXIX

Preparation of3-bromo/chloromethyl-4-carboxy-7β-cyclopentylideneammonio-3-cephemchloride from3-bromo/chloromethyl-4-carboxy-7β-isopropylideneammonio-3-cephemchloride.

In a centrifuge-tube a small amount of3-bromo/-chloromethyl-4-carboxy-7β-isopropylideneammonio-3-cephemchloride was dissolved in a small amount of concentrated hydrogenchloride. Cyclopentanone was added to the solution. After standing forthree hours the precipitate formed was filtered and washed with a littlecyclopentanone and with ether, giving white crystalline3-bromo/chloromethyl-4-carboxy-7β-cyclopentylideneammonio-3-cephemchloride.

IR (KBr-disc, values in cm⁻¹): about 3400, 1795, 1701, 1680, 1630, 1501,1403, 1350, 1094, 1059, 715, 688.

EXAMPLE XXXX

Preparation of3-bromo/chloromethyl-4-carboxy-7β-cyclohexylideneammonio-3-cephemchloride from3-bromo/chloromethyl-4-carboxy-7β-isopropylideneammonio-3-cephemchloride.

In a centrifuge-tube a small amount of3-bromo/-chloromethyl-4-carboxy-7β-isopropylideneammonio-3-cephemchloride was dissolved in a 1:1 mixture of trifluoroacetic acid andconcentrated hydrogen bromide.

Then about 1/4 volume of cyclohexanone was added. The mixture wasstirred and crystals were formed slowly. After standing for one hour thecrystalline product was filtered and washed with ether.

After drying in vacuo crystalline3-bromo/chloromethyl-4-carboxy-7β-cyclohexylideneammonio-3-cephemchloride was obtained.

IR (KBr-disc, values in cm⁻¹): about 3410, 1798, 1710, 1656, 1620, 1512,about 1398, about 1342, 1220, 1180, 1090, 1058, 693, about 620.

EXAMPLE XXXXI

Preparation of3-bromo/chloromethyl-4-carboxy-7-cyclohexylideneammonio-3-cephem bromidefrom 3-bromo/chloromethyl-4-carboxy-7β-cyclopentylideneammonio-3-cephemchloride.

7.5 g of3-bromo/chloromethyl-4-carboxy-7β-cyclopentylideneammonio-3-cephemchloride were dissolved in 20 ml acetonitrile. 20 ml of concentratedhydrogen bromide (47%) were added and for some minutes the mixture washeated to about 55° C. After a clear solution has been obtained, themixture was cooled down to 0° C. Then 10 ml of cyclohexanone was added.The mixture was red-brown and after a few minutes some crystals wereformed.

After a night standing in the refrigerator the product was filtered,washed with acetonitrile and dried in vacuo. The yield of the creamywhite crystalline3-bromo/chloromethyl-4-carboxy-7β-cyclohexylideneammonio-3-cephemchloride was 6.68 g (purity 67.6% according to NMR-assay). The productcontained also 11.6% 3-chloromethyl and some starting material as wasfound with NMR.

IR (KBr-disc, values in cm⁻¹): about 3410, 1799, 1710, 1655, 1620, 1512,about 1398, about 1340, 1220, 1180, 1089, 1057, 696, about 615.

PMR-spectrum of the bromomethyl compound (360 MHz, CF₃ COOD, δ-values inppm, int. ref. maleic acid, 25° C.): 1.64 (m, 2H); 1.93 (m, 4H); 2.79(m, 4H); 4.27, 4.34 (AB-q, J=10.5 Hz, 2H); 5.34 (d, J=4.5 Hz, 1H); 5.88(d, J=4.5 Hz, 1H).

PMR-spectrum (ibidem) of the chloromethyl compound: 1.64 (m, 2H); 1.93(m, 4H); 2.79 (m, 4H); 4.39, 4.45 (AB-q, J=12.6 Hz, 2H); 5.34 (d, J=4.5Hz, 1H); 5.91 (d, J=4.5 Hz, 1H).

EXAMPLE XXXXII

Preparation of3-bromo/chloromethyl-4-carboxy-7β-cycloheptylideneammonio-3-cephembromide from3-bromo/chloromethyl-4-carboxy-7β-cyclopentylideneammonio-3-cephemchloride.

75 mg of3-bromo/chloromethyl-4-carboxy-7β-cyclopentylideneammonio-3-cephemchloride was dissolved in 0.2 ml acetonitrile. 0.2 ml of concentratedhydrogen bromide (47%) was added. This mixture was shortly heated till aclear solution was obtained. 0.1 ml of cycloheptanone was added to theyellow solution. Slowly, but faster than in example XXXIV crystals wereformed. The solution was a little heated to accelerate thecrystallisation.

After standing for a week-end in the refrigerator the product wasfiltered off and washed with acetonitrile and ether. After drying in anitrogen-stream the yield was 61.0 mg of a white3-bromo/chloromethyl-4-carboxy-7β-cycloheptylideneammonio-3-cephembromide (purity 74.6% according to NMR-assay). The product containedalso 11.7% of the 3-chloromethyl compound and some starting material.

IR (KBr-disc, values in cm⁻¹): about 3405, 1795, 1710, 1638, about 1628,1508, about 1395, 1340, 1217, 1088, 1056.

PMR-spectrum (360 MHz; CF₃ COOD, δ-values in ppm, int. ref. maleic acid,25° C.) of the bromomethyl compound: 1.56 (m, 4H); 1.81 (m, 4H); 3.02(m, 4H); 4.27, 4.34 (AB-q, J=10.7 Hz, 2H); 5.35 (d, J=4.5 Hz, 1H); 5.80(d, J=4.5 Hz, 1H).

PMR-spectrum (ibidem) of the chloromethyl compound: 1.56 (m, 4H); 1.81(m, 4H); 3.02 (m, 4H); 4.39, 4.44 (AB-q, J=12.6 Hz, 2H); 5.35 (d, J=4.5Hz, 1H); 5.83 (d, J=4.5 Hz, 1H).

EXAMPLE XXXXIII

Preparation of3-bromo/chloromethyl-4-carboxy-7β-cyclopentylideneammonio-3-cephembromide from3-bromo/chloromethyl-4-carboxy-7β-cyclopentylideneammonio-3-cephemchloride.

75 mg of3-bromo/chloromethyl-4-carboxy-7β-cyclopentylideneammonio-3-cephemchloride were dissolved in 0.2 ml acetonitrile. 0.2 ml of concentratedhydrogen bromide (47%) was added. This mixture was shortly heated tilleverything was dissolved. The mixture was heated till clearness and verywell stirred. The yellow solution was shaken in an ultra-sonic bath andbecame then somewhat turbid. Slowly some crystals were formed and 0.2 mlof acetonitrile and 0.2 ml of cyclopentanone were added.

After standing in the refrigerator for a week-end the product wasfiltered and washed with acetonitrile and ether. After drying in vacuothe yield was 38.9 mg of3-bromo/chloromethyl-4-carboxy-7β-cyclopentylideneammonio-3-cephembromide with a purity of 83.3%.

IR (KBr-disc, values in cm⁻¹): about 3400, 1791, 1705, 1673, 1622, 1491,1392, 1342, 1217, 1089, 1056, 709, about 620.

PMR-spectrum (360 MHz; CF₃ COOD, δ-values in ppm, 25° C., int. ref.maleic acid): 1.96 (m, 4H); 2.95 (m, 4H); 4.30 (s, 2H); 5.30 (d, J=4.5Hz, 1H); 5.70 (d, J=4.5 Hz, 1H).

We claim:
 1. Halo-substituted cephalosporins of the formula IC andnon-toxic, pharmaceutically acceptable salts and esters thereof##STR36## wherein X is halogen and R₄ and R₅ are individually alkyl of 1to 8 carbon atoms or taken together with the carbon atom to which theyare attached form a cycloalkylidiene of up to 8 carbon atoms.
 2. Acompound of claim 1 wherein R₄ is methyl and R₅ is methyl or propyl orR₅ and R₆ together with the carbon atom to which they are attached areselected from the group consisting of cyclopentylidene, cyclohexylideneand cycloheptylidene and X is bromine or chlorine.